Introduction
The 3rd “Melanoma Research Bridge” meeting was held in Naples on December 2 to 4th, 2012 (Figure
Faculty and some attendants of the Bridge meeting in Naples
Faculty and some attendants of the Bridge meeting in Naples.
The most recent version of the AJCC (7th Edition) recommended including mitotic rate into the staging system as independent prognostic factor. The change was approved by the UICC. However, histopathological characteristics cannot always accurately predict who will relapse and who will remain disease free. Therefore, additional prognostic and predictive markers to determine the potential for metastatic relapse at the time of diagnosis and to guide therapeutic decisions in adjuvant settings even in early stage melanoma patients are urgently needed. Recently, a new molecular classification of melanoma is evolving based on chromosomal aberrations, gene mutations and signaling pathways activation that underlies biologically distinct subsets of melanoma requiring different clinical management. These approaches have already been proven successful in development of novel molecular diagnostics and importantly novel therapy approaches for melanoma patients.
Melanoma has historically been refractive to chemotherapy which provided very low response rates and little to no benefit in overall survival (OS). The meta-analysis of different Phase II Cooperative Group trials in metastatic Stage IV melanoma showed a median survival time of 6.2 months, 25.5% of the patients alive at 1 year, and a median progression free survival (PFS) of 1.7 months
Identification of oncogenic mutations in serine/threonine (Ser/Thr) kinase BRAF resulting in valine to glutamine substitution at codon 600 (V600E) in cutaneous melanoma led to development of an effective inhibitors and clinical trials with vemurafenib
Two antibodies that block cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) augmenting antitumor immunity, have been evaluated in phase III clinical trials i.e., ipilimumab (Bristol-Myers Squibb) and tremelimumab (MedImmune, Inc.). Ipilimumab, compared with vaccine therapy or placebo, showed to improve overall survival (OS) of metastatic melanoma patients, with a less impact on responses and PFS. Another promising immunotherapy strategy in melanoma is targeting programmed cell death-1 (PD1) receptor on activated T cells or its ligand PD-L1. Despite improvement in clinical responses with these agents only a subset of patients benefit from CTLA-4 blockade. To better control the response to these drugs new parameters of response, the immune related response criteria, an evolution of the RECIST criteria, and new parameters of management of the immune-related adverse events (irAEs) or adverse events of special interest (AEOSI) are required. Furthermore, biomarkers that can predict response to immunotherapy are much needed tools to help guide treatment decisions for these patients. Thus the emerging era of a personalized approach to the management of melanoma patients will require the identification of the specific subset of melanoma based on the driving mutations or immune response-based biomarkers to design the best drug combination for the individual patient.
Molecular advances and biomarkers
There is a great need to accurately establish diagnosis, prognosis and to define the outcome of individual melanoma patients but the existing clinicopathologic prognostic factors are not always adequate. In addition, predictive markers to determine the efficacy of treatment at the time of diagnosis and to guide therapeutic decisions for individual patients in adjuvant settings even in early stage melanoma patients emerge as an integral part of clinical management in melanoma. The focus of this section was on emerging prognostic and predictive biomarkers for melanoma as well as novel approaches providing increased opportunities for clinical application of individual markers or multimarker assays including molecular profiling, immune monitoring and functional multiparameteric assay.
Melanoma-associated antigen A3 (MAGE-A3) is a member of MAGE cancer-testis multigene family is melanoma-specific but is not expressed in normal cells. The specific expression of MAGE-A3 on various tumors provides the opportunity for a specific targeted therapy such as vaccines or adoptive T cell therapy and it serves as an eligibility factor for such treatments as only patients with melanoma expressing the antigen (MAGE-A3+) will be selected for treatment. In addition, the specific expression of MAGE-A3 gives the potential for limited off-target effects and no immune tolerance. MAGE-A3 antigen is present in major tumor types, e.g., melanoma up to 76%, multiple myeloma 60%, head and neck cancer 49%, lung cancer 35%, and thus potentially multiple therapeutic indications exists for these tumors including vaccines or T cell adoptive therapy targeting MAGE-A3
As demonstrated by pre-clinical research the MAGE-A3 protein is weakly immunogenic by itself, but formulation with various immunostimulant is able to induce a stronger immune response leading to improved protection against tumour growth in selected preclinical models
Gene expression profiling using Affymetrix HG-U133 Plus 2.0 microarray platform was used to identify a signature predictive to the clinical benefit of the MAGE-A3 vaccine treatment using pre-treatment tumor biopsies. A gene signature classifier based on differentially expressed genes was identified. In fact, overall survival in patients stratified by gene signature (GS) positivity was better than overall survival in the non-responder subgroup. Canonical pathway analysis of relevant genes identified immune related signature including genes for antigen presentation, protein ubiquitination and Interferon (IFN) signalling suggesting that the presence of immune effector cells in the tumor microenvironment predicts clinical benefit in response to treatment with MAGE-A3 vaccine
Examples of tumor antigens similar to MAGE-A3 include other Cancer/Testis antigens such as NY-ESO-1 and frequently mutated antigen TP53 that are immunogenic only in cancer patients. Various methods are available to identify immunogenic antigens and rationally design optimally effective cancer vaccines. Serological expression cloning (SEREX) and antibody profiling of protein arrays (Seromics) are tools for the discovery of immunogenic tumor antigens. Spontaneous antibody (Ab) titers against various tumor antigens have been detected in the serum of melanoma patients, non-small cell lung cancer patients and many other cancer types, but not in healthy donor sera can also serve as tumor biomarkers. Correlation of NY-ESO-1 serum antibody with clinical course following anti-CTLA-4 treatment with ipilimumab has been also established
Active immunotherapy may present an advantage if able to induce high quality immune responses when they fail to happen spontaneously. It is very important of characterize the mechanisms leading to spontaneous immunity against tumor antigens, and spontaneous immunity can be modulated with immunotherapy (e.g., ipilimumab, and possibly vaccines). The future of immune therapy will combine vaccines that stir the immune system toward the specific tumor antigen with nonspecific modulators of immune suppression such as anti-CTLA-4 or anti-PD1, as well as other drugs that broadly stimulate an array of immune cells including antigen-presenting cells (APCs). One promising group of immunomodulators is the Toll like receptors (TLR) agonists such as Cytosine phosphate Guanine (CpG) oligonucleotides that has strong immunostimulatory properties.
To inform on antigen heterogeneity and local responsive/suppressive environment, it will be very important to measure immune biomarkers in the periphery. These multiple sources of heterogeneity suggest that the therapy should be consistent with testing of tumor phenotype and specific antigen expression in individual patients as well as their immune profile. Novel approaches for detection of circulating tumor cells, circulating DNA, exosomes are explored to detect phenotypic characteristic of tumor cells that express specific combination of surface markers, driver mutations or tumor derived products as well as for immune-response monitoring.
Expression of a subset of chemokine genes is associated with presence of CD8+ T cells in melanoma metastases, so that is possible to identify patients with clinical benefit from vaccines. In fact, the “inflamed” tumor gene expression profile also may be associated with clinical benefit to ipilimumab. Two broad categories of melanoma metastases can be selected, according to gene expression profiling and confirmatory assays identifying a T cell “poor” and a T cell “rich” group. The possibility that a T cell-inflamed tumor microenvironment may be a predictive biomarker for clinical benefit from vaccination is being tested prospectively in the GSK-Bio MAGE-A3 vaccine trials. Innate immune “sensing” of tumors appears to occur via an endoplasmic reticulum-associated molecule referred to as STING (stimulator of IFN genes)-dependent pathway and host type I IFN response. “Inflamed” tumors likely are not rejected due to dominant immune suppressive mechanisms, including indoleamine 2, 3, dioxygenase (IDO), PD-L1, T regulatory cells (Tregs), and T cell-intrinsic anergy. Importantly, all of these are being targeted therapeutically in early phase clinical trials. Combinatorial blockade of selected inhibitory pathways is therapeutically synergistic in preclinical models
Three drugs are currently approved for immunotherapy in melanoma: Interferon is the only approved agent for the adjuvant therapy of melanoma, while ipilimumab is approved for metastatic melanoma as first and second line in US and second line in Europe. Interleukin-2 (IL-2) is approved for metastatic melanoma but is not currently in use in Europe. The mechanism by which IFN exerts an antitumor effect has long been debated. The immunomodulatory role of IFN is unclear, but it modulates the immune response, and has anti-proliferative, anti-vascular and pro-apoptotic effects. Type I IFNs (alpha and beta) promote proliferation and clonal expansion of CD4 and CD8 T cells, enhance antibody production of B cells, increase cytotoxic activity of natural killer cells (NK) and CD8 T cells and have negative effects on the activation and proliferation of Tregs.
Attempts to identify patients who benefit from adjuvant treatment with IFN have been undertaken almost from the point of the discovery of IFN’s benefit but to date the results of these efforts have largely been disappointing. The identification of predictive markers that permit selection of patients who are most likely to benefit would allow us to avoid the toxicity of treatment, in more than half of patients who are now offered this therapy. As emerged from meta-analyses, subgroup analyses of randomized trials and translational research studies pre-selection of patients is still controversial. The Wheatley meta-analysis of 2007
Most randomized controlled trials evaluating adjuvant therapy with IFN used Breslow thickness and lymph node invasion for staging. Subgroup analyses in randomized controlled trials have been performed by stratification according to clinical and/or pathological features at randomization, not identical over time
Studies have been performed in tissue and peripheral blood (DNA and serum) of patients receiving adjuvant or neo-adjuvant treatment with IFN to evaluate immune response correlating with clinical outcome. As an example, 20 patients with palpable lymph node metastases (AJCC stage IIIB and C) participated in a neo-adjuvant study
Additional study in an effort to understand the effects of HDI in relation to the balance of phosphorylated signal transducer and activator of transcription (STAT) pSTAT1 and pSTAT3 have been undertaken. STAT1 and STAT3 were evaluated jointly as mediators of IFN-α effects in the setting of the prospective neo-adjuvant trial
Serum multiplexed immunobead-based cytokine profiling can be used to detect melanoma cells and select patients who may be more susceptible to IFN. Powerful high-throughput multiplex immunobead assay technology (xMAP, Luminex Corp.) was used to simultaneously test 29 cytokines, chemokines, angiogenic as well as growth factors, and soluble receptors in 179 patients with high melanoma and 378 healthy individuals, participating in E1694 study. IFN treatment was correlated with decreases in levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and hepatocyte growth factor (HGF) and increased levels of anti-angiogenic IFN-γ inducible protein 10 (IP-10) and IFN-α. Pretreatment levels of IL-1α, IL-1β, IL-6, TNF-α, and chemokines MIP-1α and MIP-1β were found to be significantly higher in the serum of patients with longer RFS values
Methylthioadenosine phosphorylase (MTAP) a house keeping gene is constitutively expressed in most normal cells and tissues. Loss of MTAP activity was related to deletions in human chromosome 9p21, encoding the tumor suppressor genes CDKN2A and CDKN2B, MTAP and IFN alpha and beta and to epigenetic regulation by promoter hypermethylation. In malignant melanomas, selective deletions in this chromosomal region or promoter hypermethylation are known to result in a loss of MTAP protein expression. MTAP expression has significant impact on STAT1 activity. Using tissue microarrays assembling 465 nevi, primary melanomas and metastases, the expression of MTAP was investigated. In subgroup analysis of patients with tumor thickness of 1.5-4.0 mm revealed a significant survival benefit from adjuvant IFN treatment regarding RFS (p < 0.05) if MTAP expression was observed in the primary melanoma. Patients with STAT1 positive melanomas also tended to benefit from IFN concerning RFS (p = 0.074) and showed a significant benefit of OS (p < 0.05)
Peripheral blood lymphocytes (PBL) from melanoma patients have reduced phosphorylation of STAT1 upon IFN-α stimulation, demonstrating a defect in Type I IFN signaling. Such defects could be partially restored by prolonged stimulation with IFN. Archived peripheral blood mononuclear cells (PBMCs) from 14 Stage IIIB-C melanoma patients pre and post treatment were analyzed for STAT1-Y701 phosphorylation (pSTAT1) levels by phospho-flow cytometry. Significant increase in STAT1 activation in peripheral blood T cells, but not B cells, upon IFN-stimulation was evidenced from Day 0 to Day 29. Moreover this increase of pSTAT1 in peripheral blood T cells also correlated with good clinical outcome. Between patients who showed increased pSTAT1 signaling after HDI therapy, only those who displayed modest augmentation had good outcome.
A multi-factorial genetic model for prognostic assessment of high risk melanoma patients receiving adjuvant IFN has been performed, analyzing data of 284 melanoma patients. In univariate analysis of five-marker genotyping signature was prognostic for melanoma overall survival. This signature defines high and low risk groups and it was shown to be an independent predictor of OS, after controlling for stage
Specific human leukocyte antigen (HLA) class I and II antigens (eg., HLA-A1, HLA-A11, HLA-Cw7, and HLA-DQ1), have previously shown an association with response to interferon therapy or overall survival of patients with metastatic melanoma. A total of 284 high-risk melanoma patients participating in a randomized trial and 246 healthy controls were molecularly typed for HLA class I and II
In conclusion, the selection of patients for IFN therapy should consider parameters as ulcerated primary melanoma which will be prospectivly validated in EORTC 18081 trial, evaluation of level of biomarkers such as STAT1 ↑, STAT3 ↓, pretreatment levels of proinflammatory cytokines, and patients with HLA Cw*06 polymorphism.
Several polymorphisms have been found within the CTLA-4 gene were shown to have an association with the development of autoimmune disease as Graves’s disease, type 1 diabetes and Addison’s disease. Specific CTLA-4 polymorphisms have previously shown an association with autoimmune symptoms and response to ipilimumab (i.e., GG allele of JO30). GG is associated with decreased expression of CTLA-4 upon T-cell activation and thus a higher proliferation of T-cells. Cohort of 286 melanoma patients treated with high-dose adjuvant IFN in a randomized trial and 288 healthy controls were genotyped for six CTLA-4 polymorphisms previously suggested to be important, AG 49, CT 318, CT 60, JO 27, JO30 and JO 31
Ipilimumab, an anti CTLA-4 specific monoclonal antibody therapy, results in a durable clinical benefit for a subset of patients with refractory melanoma and has reversible mechanism-based side effects. Though, definite correlates of clinical response are not established several potential biomarkers of response positively correlate with improved clinical outcome following ipilimumab therapy. The absolute lymphocyte count (ALC) in patients who achieved clinical benefit from ipilimumab had a greater mean increase in ALC after starting therapy than patients who had progressive disease. In addition, to efforts monitoring T cell subpopulations during treatment with CTLA-4 blockade, characterization of antigen specific antibody and T cell responses has similarly led to associations between immunologic changes and benefit from CTLA-4 therapy. Serological studies have evaluated antibody responses against a number of tumor associated antigens, including MAGE, Melan-A, MART-1, gp-100, and Tyrosinase. Patients, who had detectable humoral responses against the cancer-testis antigen, NY-ESO-1 were more likely to experience clinical benefit than those with negative antibody titer.
Gene expression analysis of flow-cytometry purified CD4+ and CD8+ T cells was employed to assess gene profiling changes induced by ipilimumab. Selected molecules were further investigated by flow cytometry on pre, 3-month and 6-month post-treatment specimens. Ipilimumab up-regulated Ki67 and ICOS on CD4+ and CD8+ cells at both 3- and 6-month post ipilimumab (p ≤ 0.001), decreased CCR7 and CD25 on CD8+ at 3-month post ipilimumab (p ≤ 0.02), and increased transcription factor Gata3 in CD4+ and CD8+ cells at 6-month post ipilimumab (p ≤ 0.001). Increased EOMES+CD8+, GranzymeB+ EOMES+CD8+ and decreased Ki67+EOMES+CD4+ T cells at 6 months were significantly associated with relapse (all (p ≤ 0.03). Decreased Ki67+CD8+ T cells were significantly associated with the development of irAE (p = 0.02). At baseline, low Ki67+EOMES+CD8+ T cells were associated with relapse (p ≤ 0.001), and low Ki67+EOMES+CD4+ T cells were associated with irAE ((p ≤ 0.008). Up-regulation of proliferation and activation signals in CD4+ and CD8+ T cells were pharmacodynamic markers for ipilimumab. Ki67+EOMES+CD8+ and Ki67+EOMES+CD4+ T cells at baseline merit further testing as biomarkers associated with outcome and irAEs, respectively
A systems-level approach is required for comprehensive understanding of the interconnected components, pathways, and cell types associated with an immune response. Single cell network profiling (SCNP), is a novel platform for assessing and measuring immune function/dysfunction at a “systems” level. SCNP is a multiparametric flow-cytometry based analysis that can simultaneously measure, at the single cell level, both extracellular surface markers and changes in intracellular signaling proteins in response to extracellular modulators
When applied to pathways shown to be important in disease pathology, this method of mapping signaling networks has potential applications in the development of disease profiling, the identification of novel disease targets, predictive tests for therapeutic response and patient selection and overall for improved efficiency of drug development
Better methods to determine melanoma progression would allow further improvements in the prognostic assessment of melanoma patients. Importantly, the accurate prognosis will benefit proper risk stratification of the early stage melanoma patients for adjuvant treatment. The diagnosis of primary cutaneous melanoma at the pathological level can represent a daunting task. Several features are used to diagnose melanoma, such as cytologic atypia, maturation with descent, poor circumscription, presence of mitoses, and asymmetry. However, the pathologic diagnosis of melanoma remains challenging, resulting in a high degree of inter-observer variability Although traditional cancer diagnostics approaches including histopathology and IHC will likely remain standard tools for the future the upcoming molecular analyses might be incorporated in the context of these established methods when the definite diagnosis cannot be reached. They potentially might be systematically incorporated and lead to improvement of the AJCC/IUCC system.
Different approaches are routinely explored for discovery and validation of biomarkers to improve diagnosis, classification and prognosis. Gene expression profiling of a series of freshly acquired nevi, primary, and metastatic melanomas identified differentially expressed gene sets during melanoma progression, including nevus to primary melanoma, radial versus vertical growth phase melanoma or primary
A three-marker IHC-based prognostic assay in primary cutaneous melanoma developed and tested in a cohort of 395 patients. The markers evaluated included NCOA3 (nuclear receptor coactivator 3), RGS1 (regulator of G protein signaling), and SPP1 (osteopontin), and were scored both by a pathologist blinded to patient outcomes on a 0–3 scale, and using a digital imaging analysis. The outcome parameters evaluated were disease-specific survival (DSS, primary endpoint) & sentinel lymph node (SLN) metastasis. Each marker alone had been shown to significantly predict DSS and SLN status. Marker expression was then validated in an independent cohort of 141 cases from German Cancer Registry (Heidelberg/Kiel cohort). The multi-marker score was independently predictive of DSS in cohorts, surpassing tumor thickness and ulceration. Ulceration, an established prognostification factor for localized primary melanoma that has been included to the AJCC synoptic for microstaging of melanoma. Thus, the multi-marker assay can be considered a very important prognostic factor to predict DSS and SLN metastasis
More recent studies have focused on the role of the pleckstrin homology domain-interacting protein (PHIP) gene in melanoma progression. PHIP emerged as the gene most highly expressed in melanoma metastases
Based on recent analyses, PHIP can be considered a novel marker and mediator of melanoma metastasis. shRNA-mediated targeting of PHIP significantly reduced murine and human melanoma invasion and metastasis. PHIP mediated its pro-invasive effects by virtue of activating AKT. The prognostic role for PHIP in human melanoma was demonstrated in a study analyzing a cohort of 345 cases. There was a significant association between PHIP levels and ulceration.
Genotypic analysis aimed to identify the molecular subtypes of melanoma demonstrating activated PHIP, based in part on its activation of AKT. AKT activation in melanoma is associated with NRAS mutation or BRAF mutation and PTEN loss. It was hypothesized that if PHIP expression is sufficient for AKT activation, then high PHIP-expressing melanomas should not have mutant NRAS or BRAF/PTEN mutants. The great majority of high PHIP-expressing melanomas were devoid of NRAS or double BRAF/PTEN mutants. Thus, PHIP levels are enriched in triple negative (WT BRAF, NRAS, and PTEN) and double negative melanoma (mutant BRAF and WT NRAS and PTEN).
The PHIP gene normally resides on the 6q14 locus, but in melanoma 6q loss has been reported. By fluorescent in situ hybridization (FISH) analysis, not only is the PHIP locus preserved in melanomas, approximately 80% of high PHIP-expressing melanomas had an increased copy number. More recent analyses were presented indicating a prognostic role for high PHIP copy number. Data were presented from experimental models of melanoma showing that suppression of PHIP results in reduced glycolysis, lower VEGF levels and decreased tumor angiogenesis. On the basis of these results, a model of ulceration development was presented in melanoma in which PHIP activation results in activation of the IGF1R pathway and AKT, ultimately resulting in increased glycolysis, invasiveness, and angiogenesis, resulting in increased capacity to develop both ulceration and metastasis.
Between the mechanisms of acquired (late) BRAF inhibitors (BRAFi) resistance, there is a predominance of MAPK-reactivation. This can happen by RAS mutations, MEK mutations, BRAF V600E/K splicing or amplification. This phenomenon reflects the fact that effective initial on-target MAPK pathway inhibition in the majority of melanomas provides the selective pressure for the tumors to re-activate a melanoma-addicted pathway. By whole-exome sequencing, AKT gain of function mutations were identified in a minority of melanoma with acquired BRAF inhibitor resistance. These AKT mutants conferred BRAF inhibitor resistance most potently in a cells which only weakly upregulated AKT in response to BRAF inhibition. Thus, AKT mutants amplify an adaptive response to BRAF inhibition. This adaptive AKT rebound in response to BRAFi is widespread early during therapy. The landscape of acquired resistance is quite heterogeneous but core pathways exist. In a single patient, both MAPK- and AKT-dependent mechanisms of acquired resistance can concur.
Combination therapies
Combination therapies are the most promising approach to add the therapeutic value to therapy with a single drug. A major challenge for the combination treatment in melanoma is to overcome intrinsic and acquired resistance. The elucidation of signaling pathways underlying resistance is important in order to develop effective personalized targeted strategies for individual patients’ as multiple mechanisms or resistance develop during the initial treatment. Also, microenvironmental factors and immune response need to be considered in designing appropriate treatment strategies and developing markers predictive of response. In addition, the efficacy of the treatment could depend on using sequential of combined modalities. Finally, preclinical models and methods concerning the profiling of individual tumors need further improvement. Despite, these numerous challenges several combination therapies for melanoma entered clinical evaluation, as discussed during this session.
The prognosis of melanoma varies widely by stage and for high risk surgically resected melanoma (AJCC stage IIB and higher) the risk of recurrence and death exceeds 35-40% at 5 years. On the other hand, most of cases are diagnosed at early stage and in this setting patients with high risk of developing metastatic disease may benefit from adjuvant therapy which presents the best opportunity at curing melanoma at this time. Advanced melanoma displays immunological tolerance and it is hypothesized that the role of immunotherapy in the adjuvant setting could be the greatest before immune tolerance is established. Multiple clinical trials have been performed with adjuvant IFN-α immunotherapy for resected melanoma, and 3 meta-analyses reviewing these trials have been reported to date. The Mocellin meta-analysis of 14 trials reported 18% reduction in the risk of relapse and 12% reduction in the risk of death with the use of adjuvant IFN overall
While most IFN adjuvant trials in melanoma have reported a RFS benefit, a significant impact on OS has been reported only in the high dose IFN regimen as tested in the E1684 trial. E1684 randomized high risk melanoma patients after surgery to either observation or treatment with HDI given intravenously, 5 days a week for 4 weeks (induction) then subcutaneously at lower dosage, 3 days a week, for 48 weeks (maintenance). E1694 randomized patients to be treated with adjuvant ganglioside GM2-KLH/QS-21 (GMK) vaccine for 96 weeks versus HDI. HDI demonstrated significant improvements on RFS and OS in the E1684 (versus observation) and E1694 (versus GMK). In E1684, at the median follow up of 6.6 years, compared to observation, there was a 33% reduction in the hazard of death in favor of IFN 39% reduction in the hazard of recurrence and the greater benefit was seen in patients with a high tumor burden (N1-2b). In E1694, compared to the GMK vaccine, there was 24% reduction in the hazard of mortality and 25% reduction in the hazard of relapse, with the greatest benefit seen in patients with a lower tumor burden (T4, N-)
The EORTC 18991 trial hypothesized that prolonged treatment with IFNα-2b is needed to obtain a maximal anti-angiogenic effect, and thus has compared observation with an intended 5 years of maximally tolerable doses of pegylated IFNα-2b (pFNα-2b) for patients with resected, stage III melanoma (TxN1–2 M0). During the first 8 weeks, pegylated IFNα-2b was administered at 6 mg/kg per week followed by 3 mg/kg per week maintenance therapy for up to 5 years. The study was designed to measure changes in distant-metastasis-free survival (DMFS) as the primary endpoint, and was powered to detect a 9.75% absolute difference in DMFS at 4 years. The study results were first reported in 2007 at a median follow up of 3.8 years and later updated in 2011 at a median follow up of 7.6 years
Predictive markers of therapeutic benefit are needed to accelerate progress in the adjuvant therapy of melanoma to treat only those who would relapse and to treat only those who have the capacity to respond. Biomarkers could serve to select patients who would benefit from treatment with IFN and to spare side effects of the treatment for patients who are not likely to respond. Biomarkers of risk & benefit that have been evaluated in studies of adjuvant IFN include S100B, autoimmunity, the proinflammatory cytokine profile in serum. Autoimmunity was identified to be a predictive factor for response in adjuvant treatment with IFN as demonstrated by the presence of thyroid, anti-cardiolipins, anti-nuclear, and anti-DNA autoantibodies
Recently, it has been postulated that the effects of IFN could be even related to the BRAF mutational status in melanoma (Soldano Ferrone, unpublished). BRAF V600E mutation-activated pathway is present in approximately half of cutaneous melanomas. Although, BRAF inhibitors induce rapid anti-tumor responses in about 50% of patients many challenges still exist to optimize BRAFi-based therapy. First, about 40% of patients do not respond to BRAFi due to primary resistance. Second, only about 5% of patients have complete responses. Third, the median duration of response among responders is less than 7 months (secondary resistance). These clinical findings emphasize the need to design rational combinatorial therapeutic strategies to increase the frequency of complete responses and to improve the duration of response to BRAFi therapy. In this regard we are testing the hypothesis that the BRAFi and type I IFN-α combination is more effective than the individual agents in suppressing human melanoma cell growth both
The interaction of type I IFN with target cells is mediated through a heterodimeric receptor composed of two subunits referred to as IFNAR1 and IFNAR2 with the binding of this receptor playing a key role in the antiproliferative activity of type I IFN. The available evidence in the literature argues for an IFNAR1 down regulation in melanoma cells harboring a mutated active BRAF
Another effect of this combination is the upregulation of HLA Class I molecules as well as of antigen processing machinery (APM) for melanoma antigens, inducing a greater sensitivity to T cell recognition which supports the rationale for the association with other immunotherapies such as anti-CTLA-4 treatment. In a Phase II Trial, tremelimumab was tested in combination with high-dose IFN-α yielded a high overall response rate. Overall, 37 patients were treated with tremelimumab given at 15 mg/kg every 90 days, and high-dose IFN-α simultaneously given at standard FDA-approved doses
Various studies of combined treatment of chemotherapy and cytokines have been conducted with contradictory results. The phase II E4697 adjuvant trial for high risk resected stage III-IV melanoma tested the hypothesis that granulocyte macrophage colony-stimulating factor (GM-CSF) and/or Tyrosinase peptide-based vaccine would be of therapeutic benefit. However, the trial showed that neither GM-CSF nor peptide vaccination achieved the objectives of significant improvement in OS or DFS. A suggestion of a favorable effect of GM-CSF on DFS and OS among Stage IV subjects was seen at subset analysis that warrants further investigation
Other adjuvant modalities evaluated were cellular vaccines: Canvaxin (was detrimental in Ph III trials), MAGE-A3 peptide vaccination given in adjuvant with a TLR-9 agonist (results are pending), and anti-CTLA-4 blocking monoclonal antibody therapy with ipilimumab (EORTC 18071 and US Intergroup E1609 trials are still pending). Adjuvant trials of BRAFi and BRAFi plus MEKi are ongoing. EORTC 18071 trial randomized patients with resected stage IIIA, IIIB, IIIC melanoma, to receive adjuvant ipilimumab at 10 mg/kg or placebo. Intergroup E1609 Adjuvant Phase III trial randomizes patients with resected stage IIIB, IIIC, M1a or M1b to be treated with ipilimumab at 10 mg/kg or 3 mg/kg versus HDI.
The induction of an effective T cell mediated antitumor response is dependent upon the encounter of cytotoxic T-cells with their cognate antigen. However, the identification of tumor antigens that can successfully mediate an effective antitumor response with ACT immunotherapy requires more research. In mice, the adoptive transfer of T cells specific for a nonmutated melanoma differentiation antigen (gp100) can mediate tumor regression of established B16 murine melanoma
Adoptive cell transfer (ACT) immunotherapy proves the concept of effective antitumor T cell mediated cytotoxicity and is the most effective form of immunotherapy for patients with metastatic melanoma. Current ACT clinical protocols using autologous TILs can mediate durable complete responses in 22% of patients with 95% of patients surviving beyond 3 years
BRAF mutations have been identified as the most frequent in melanoma and development of BRAF inhibitors for clinical setting has been extremely successful. Vemurafenib is a “selective” BRAF Inhibitor of V600 mutant BRAF kinase in melanoma, with an optimal activity at the dose of 960 mg twice daily (bid), as defined by the phase I, II and III clinical trials. Based on the Phase III trial comparing vemurafenib
Despite the impressive single-agent clinical activity several problems exist that need to be overcame to improve the efficacy of vemurafenib i.e., toxicity of the drug, reactivation of the MAPK pathway, development of resistance and occurrence of the secondary tumors. Combination regimens based on mechanisms of resistance and/or activation of oncogenic pathways that involve other therapeutic targeting agents for melanoma (MEK, PI3K, AKT, CDK4/6, Hsp90 etc.), are expected to have additive or synergistic effects on clinical outcome when added to BRAF inhibitors. Other promising avenues of melanoma drug development include targeted to the host i.e., immunotherapy (ipilimumab, anti-PDL1, pIFN). Another, possibility is anti-angiogenic therapy (e.g., bevacizumab and IL-2) in combination with targeted therapy (BRAF, MEK, ERK etc.) targeting the tumor.
Other tumors harboring BRAF mutations have also been identified. For example, 10% of colon cancer (CRC) population harboring BRAF mutation has extremely poor prognosis. In the Phase I trial CRC expansion cohort, activity of a single agent vemurafenib has been disappointing (1 PR out of 20 total)
However, it is virtually impossible to explore all potential drug combinations using tumor material before and after tumor progression or performing sequential biopsies from the patients considering functional complexity of the tumor and developing resistance. Model systems to identify novel combination treatment for cancer are lacking. Possible solutions are: to develop
As with all tumors, clinical trials for melanoma are not consistently conducted with a solid rationale from preclinical animal efficacy studies. All preclinical models have limitations and often fail to predict antitumor efficacy in human clinical trials. Mouse models either xenotransplanted or genetically-engineered completely mimicking human melanoma biology are not available and the use of immunodeficient hosts that do not allow to address the effect of the immune system account for the existing translational “bench to bedside” gap. New models are required to develop predictors of response and to establish combination molecular or/and immune-based therapies since some combinations may be antagonistic or affect tumor infiltrating lymphocytes (TIL) effector function.
Maximum BRAF V600E level reduction occurs during Cycle 2 and 3 in vemurafenib treated patients as measured using RT-PCR based assay
The lessons learned from BRAF inhibitor development lead us to conclusion that targeting mutated BRAF gene in melanoma is a valid strategy, but not sufficient to improve overall survival. Careful biochemical and genetic analysis of the tumor samples and/or short term cultures derived from patient’s tumors including stroma and TILs may point to the useful combination treatments for individual patients. Remaining challenges include proper PK/PD investigations as part of Phase I studies of new drug candidates to truly evaluate their therapeutic potential. Importantly, correlative studies should be incorporated in the early drug development including exploration of novel targets, developing resistance, combinatorial strategy testing, and dosing strategies. Computer simulation studies of non-standard drug scheduling may also help to move beyond simple sequential or combination therapy to achieve the goal of improving patients’ outcome.
BRAF inhibitors have different effects in BRAF V600E mutant melanoma and BRAF wild type cells. Reduction of MAPK signaling and consequent block of the cell proliferation in the V600 mutated melanoma cells whereas activating MAPK signaling and progression through the cycle cycle in cells harboring wild type BRAF is observed. The evidence suggests that the use of BRAF inhibitor alone leads to reactivation of the MAPK pathway and enhances its activation through another member of RAF family of kinases CRAF. This “paradoxical” MAPK activation by BRAF inhibitors can result in the development of cutaneous squamous cell carcinoma of keratoacanthoma type (cuSCC/KAs), a typical cutaneous manifestation of BRAF inhibitor therapy such as with vemurafenib or dabrafenib in ~25% of patients
In preclinical mouse-models, cyclooxygenase 2 (COX2) inhibitors prevent the formation of squamous cell carcinomas of the skin even in COX-2 deficient mice exposed to DMBA and TPA, a commonly used skin carcinogenesis mouse model. A hypothesis based on these results is that the hyperactivation of COX-2 results in increased prostaglandin E2 (PGE2) level that activates PI3K and Src via G protein coupled receptors EP2/4. PI3K and Src activate HRAS and CRAF, and trans-activate EGFR that results in the paradoxical activation of MEK, during the treatment with vemurafenib. This was demonstrated in another murine preclinical model where COX-2 inhibition with COX-2 inhibitor (celecoxib) blocked acceleration of DMBA/TPA-induced skin tumor development by a BRAF inhibitor
As already discussed, vemurafenib was the first BRAF inhibitor that show significant clinical benefit in melanoma patients. Treatment with vemurafenib leads to a reduction in the level of phosphorylated MEK and ERK in tumors containing mutated BRAF V600E which is associated with clinical response. BRAF inhibitors can effectively inhibit 90-100% of MAPK signaling. This underlies the rapid response and clinical effect of this class of drugs on patients with BRAF V600 mutant melanoma. Importantly, it appears that the great majority of patients treated with single BRAF inhibitor will eventually have progressive disease despite successful inhibition of the V600E BRAF and high objective response early in the course of therapy. ERK (and MEK) phosphorylation, Cyclin D1, and Ki-67 are again upregulated in samples taken at the time of progressive disease as compared to their reduced expression early in therapy.
Available data suggest that the resistance to vemurafenib is not related to the development of secondary “gate-keeper” mutations of BRAF V600E gene that prevents the binding of the treatment drug to BRAF. In fact, a large number of resistant melanoma samples have had entire BRAF gene sequenced and no secondary mutation were found. Instead the resistance is mediated by reactivation of MAPK signalling in most tumors through alternative mechanism. Several mechanisms of acquired resistance to BRAF inhibition have been identified including: acquisition of NRAS and MEK1 activating mutations, upregulation of receptor tyrosine kinases (i.e., PDGFRB, ERBB2), PTEN loss, activation of the Ser/Thr MAPK kinases (COT), NF1 loss, BRAF truncations, BRAF amplification and possibly others. All of these mechanisms involve the reactivation of the MAPK pathway
Primary resistance to BRAF inhibition is present in approximately 5-10% of patients with BRAF mutant melanoma patients including CCND1 amplification in tumors, leading to downstream over expression of Cyclin D1 and enhanced CDK4 expression. CTNNB1 mutations co-occurred pre-therapy and at progression at ~5-10% frequency. Sanger Sequencing (exons 2, 3 and 6) of MEK1 demonstrate, MEK1P124 mutations co-occur at low frequency (6/132 pts, ~7%) with BRAF V600 mutations prior to treatment. Rare MEK1 mutations (codons 56, 121, and 203) that are known to reactivate MAPK pathway and be resistant to MEK1 inhibitors have been identified at progression. These might represent pre-treatment markers of resistance which can help to identify patients who are not likely to respond to BRAF inhibitors.
Recent RNA interference screen showed that neurofibromin (NF1) drives resistance to BRAF inhibition
Another novel approach to melanoma treatment under investigation is the use of oncolytic viruses (OVs). OVs are native or recombinant viral vectors that mediate host anti-tumor activity through two distinct mechanisms
An attenuated, recombinant herpes simplex type 1 virus (HSV-1) encoding GM-CSF has been constructed. This vector has demonstrated regression of both injected (oncolytic) and uninjected tumors in murine models. HSV-1 encoding human GM-CSF was constructed and has been named Talimogene Laherparepvec (T-VEC). T-VEC was shown to be safe in a Phase I clinical trial and was tested in 50 patients with locally advanced and metastatic melanoma in a Phase II study
In summary, understanding of the molecular drivers of melanoma provides opportunities to target the disease in a rational manner that may ultimately reduce the mortality from the disease. Different signaling pathways have been identified in melanoma with genomic analysis of primary melanoma giving an insight into the molecular drivers of the disease at an early stage in disease progression and include mutations in a variety of genes including well established BRAF, NRAS, KIT, MAPK kinases, and novel genes such RAC1. These genomic changes frequently involve classic oncogenic pathways including the PI3K/AKT/mTOR, RAS/RAF/MEK/ERK and the CyclinD1/CDK4/pRB pathways. Mutations in genes that activate these pathways provide opportunities to therapeutically target the disease. Mutations in BRAF in about 50% of melanomas can be inhibited by drugs that target the RAS/RAF/MEK/ERK pathway including vemurafenib, dabrafenib, and trametinib. Similarly, mutations in NRAS in about 15-18% of melanomas also activate the RAS/RAF/MEK/ERK pathway that can be inhibited by MEK-inhibitors such as MEK 162. Other therapeutic opportunities exist in targeting mutations in ERBB4 present in about 10% of melanoma patients that could be inhibited by lapatinib. Upregulated EphB4 receptor expression present in about 10% of melanomas could be inhibited by nilotinib, and imatinib, sunitinib; and nilotinib inhibit KIT mutations that occur in about 3-5% of melanomas.
BRAF inhibitors have dramatically changed the outcome of patients with advanced disease that contain mutations at the codon V600. However, the responses are not long lasting and result in progressive disease in majority of patients. Significant progress has been made in understanding the mechanisms of the intrinsic and acquired resistance that can be both MEK-dependent and MEK-independent. To overcome MEK-dependent resistance the combination of a BRAF inhibitor (dabrafenib) with a MEK inhibitor (trametinib), has resulted in significant extension of PFS, when compared to dabrafenib as single agent. BRIM 7 trial combining vemurafenib with a potent inhibitor of MEK1/2 (GDC 0973 or cobimetinib) demonstrated a high response rate in melanoma patients’ naïve to BRAF inhibitors. However, adding the MEK inhibitor to vemurafenib-progressive patients, has led to a much less impressive response rate (< 25%). The well-documented immunotherapy approaches for melanoma targeting key T cells immune check points such as CTLA-4 and PD1-PD-L1 have already validated the merits for combining these agents with targeted therapy approaches. Targeted therapy increases the sensitivity of the tumor for immunotherapy by recruiting immune cells into melanoma lesions supporting the merit of combining those two types of therapy to achieve clinical benefits. Other possible approaches to overcome developing resistance are to target other aspects of melanoma biology downstream of BRAF such as cell cycle regulation, tumor metabolism or activation of an immune response. Potential therapeutic strategies include the use of CDK4 inhibitors such as PD-0332991, and inhibitors of glycolytic regulators of tumor metabolism such as inhibitors of pyruvate dehydrogenase kinase or lactate dehydrogenase that inhibit tumor cell glucose metabolism. A cancer drug based on oncolytic virus has also succeeded in a late stage clinical trial giving another agent for multi-modality care.
In conclusion, many mechanisms of resistance have been driving progression during anti-BRAF therapy, likely involve MAP-kinase pathway in most cases, and it is likely that more than one resistant mechanism is present at the same time. It’s difficult, if not impossible, to target all the resistance mechanisms after they have been “activated”, but combination therapy used upfront may prevent resistance (i.e., BRAFi plus MEKi). Combination therapy approaches that incorporate targeted agents (e.g., vemurafenib) and immunotherapy (e.g., ipilimumab, anti-PD1/PDL1) are most promising and are already clinically explored. Despite the remaining challenges, it is certain that rational combination therapy for treatment of patients resistant to BRAF inhibitors represents the future of metastatic melanoma treatment.
Novel concepts
Melanoma is a complex and heterogeneous disease and tumor may be composed of distinct cell populations with distinct molecular features and varying response to treatment. Inter-and intra-tumor heterogeneity within primary tumours and between primary and metastatic sites as well as developing resistance suggest the need for novel approaches to identify predictive markers closer to patients’ tumor allowing for rational design of combination therapies instead of using a single biopsy assumption. Additional challenge is how to use the knowledge that cancer development and progression depends on immune system and stroma and several areas of new investigations provide promissing insights with regard to development of successful treatments. Other studies including the role of microbiota, mechanisms of rejection and metastasis process are essential for continuous progress in the treatment of melanoma. Thus, it is important that the agents to down-regulate the immune suppression, drugs targeting vasculature or crucial regulators of metastasis complimentary to targeting tumor cells are considered for rational therapeutic strategies.
Humans have co-evolved with microbial partners. We are a composite of species: including bacteria, fungi and viruses. In and on our bodies, microbial cells outnumber the human cells by about 10 fold. In the intestine, the total microbial DNA (the microbiome) may contain 100 times more genes than our ‘own’ human genome. The microbiome is an integral part of our genetic landscape and regulates metabolic functions. The development of the immune system is even dependent on interactions with the commensal microbiota.
Commensal bacteria affect local and systemic inflammation/immunity, influencing skin, mucosal and intestinal immunity, and relative inflammation. In experimental mouse model of subcutaneous transplanted tumor, antibiotics suppress the anti-tumor effect of anti-IL-10R/CpG therapy, decrease inflammatory cytokine production in the treated tumors, and suppress early necrosis of the tumor. Antibiotics treatment also impairs the anti-tumor effect of chemotherapy with platinum compounds such as oxaliplatin
Tissues respond to infection or damage with inflammation and immunity in which all tissue cell types actively participate in order to fight infections, repair damage, and restore tissue integrity and function. Chronic inflammation can promote all phases of carcinogenesis, from initial genetic alterations promoting tumor development, to establishing tumor environment that promotes tumor initiation and progression, and triggering immunosuppressive mechanisms that prevent anti-tumor immune response. This intrinsic inflammation is carried out by epithelial cells, endothelial cells (ECs), fibroblasts, infiltrating hematopoietic cells and other stromal cells that produce cytokines, chemokines and growth factors that affect tumor cells phenotype as well as create micro-environment in surrounding tissues which is responsible for activation of a proinflammatory program. By contrast, extrinsic inflammation is activated by the tissue response to the malignant cells, and it is predominantly mediated by the infiltrating inflammatory cells and regulates tissue rearrangement, angiogenesis and ability of tumor cells to disseminate
Involvement of different mechanisms and compounds in inflammation is postulated. For example, the signaling adaptor molecule myeloid differentiation factor 88 (My88) downstream of most TLRs and of the IL-1 family is central for the activation of NF-κB mediated innate inflammatory pathways. My88 is required for RAS induction of pro-inflammatory genes through a positive feed-back mediated by IL-1α binding to the MyD88-coupled IL-1R. An IL-1α autocrine loop in oncogenic RAS transformed mouse keratinocytes is required for both the secretion of pro-inflammatory mediators and, in part, for the transformation phenotype
Tumor-associated macrophages (TAM) are a key component of cancer promoting inflammation
Ulcerated melanoma is a distinct biologic entity, considering that survival is much lower for same Breslow thin melanomas
Intratumour heterogeneity (ITH) is variability within a tumour. Clinicians, particularly pathologists, have understood morphological differences in cancer for decades. ITH includes variability within primary tumours and between primary and metastatic sites
In the same patient. ITH can be viewed in an evolutionary framework and has been identified clearly in renal cell carcinoma (RCC). A fundamental question for personalised medicine is whether the putative driver identified from tissue “x” at time “y” really does drive the metastatic disease in the patient in the clinic. Furthermore, do image-guided biopsies of large renal tumours represent the entire primary, never mind the burden of metastatic disease? Single biopsy assumptions are that the tumour somatic/transcriptomic landscape is represented and provides robust prognostic and predictive biomarkers of outcome and that mutations are ubiquitously present in every region of a tumour and can be used for sequencing analysis to stratify patients for therapeutic trials. But these assumptions are not always valid. The E-PREDICT study
There may be evidence for ITH in melanoma; polyclonality of BRAF mutations in primary and metastatic samples has been shown and in samples from patients resistant to Vemurafenib, persistence of BRAF mutations has been shown. These melanomas, however, have acquired NRAS mutations or PDGFRβ overexpression. Furthermore, distinct subclones identified with IHC within a metastasis progressing on vemurafenib have been studied: one remained BRAF mutant; the other acquired NRAS mutation in addition. Re-sensitisation of patients to BRAFi therapy after time off treatment is another interesting observation that may be explained by ITH
Concluding, intratumour heterogeneity has been around for decades, and in RCC, as in other cancers, it can be demonstrated using multiple methods. Evidence for ITH in melanoma is perhaps less convincing at present, but it is plausible though that it may be relevant to drug resistance. Further study is needed (e.g. detailed longitudinal tissue collection), to investigate if ITH may impact on therapeutic strategies.
Stereotactic radiotherapy (SRT) can be considered the best local treatment for brain metastases, controlling nearly all treated lesions for at least several months. Since the advent of SRT over a decade ago, the pattern of survival has changed to longer and more likely to succumb to extracranial disease than to brain metastases. Melanoma brain metastases have always been considered a “death sentence” with less than 6 months’ median survival for most subgroups of patients. Fortunately, questions about the risks and biology of brain metastases (e.g., molecular pathogenesis, tumor-host relationships, site of primary and its environmental and genetic features) have started to see answers, and much new information is expected from collaborative networks that have been developed to answer these and other questions about biology and management of brain metastases. In 296 patients with resected brain met(s) BRAF and NRAS status was determined, and they were evaluated between Jan 2005-Dec 2011 (before BRAFi, MEKi therapies)
Ipilimumab was tested in two clinical trials to identify safety and overall survival of patients with advanced melanoma and brain metastases. Dose/schedule were identical at 10 mg/kg q 3 wks × 4, followed by 10 mg/kg q 12 weeks until relapse or intolerance. The two trials are the CA184-042 trial and the CA184-045, an open-label, expanded-access treatment protocol. Patients who qualified for these trials experienced an approximately 20%-25% durable benefit at 2+ years of follow-up, with rare progressions beyond this time.
The NIBIT trial, associating ipilimumab at the dosage of 10 mg/kg with fotemustine, treated melanoma patients with brain metastases. Results were 40% overall disease control rate, and 50% CNS disease control rate, with a good toxicity profile. The role of fotemustine is unknown and is being studied in an ongoing randomized trial but the drug is unavailable in the U.S. Although ipilimumab has been combined with cytotoxic agents such as dacarbazine and temozolomide (TMZ), the benefits of each component and their interactions could not be assessed from the trial designs. Toxicities may limit the further exploration of such combinations, and there is currently greater enthusiasm for combinations of checkpoint blockade with molecularly targeted agents. Nevertheless, some of the current immunotherapeutic strategies are investigating the use of selected chemotherapies to transiently lymphodeplete the patient in an effort to alter T cell subsets and stimulate them with increased levels of homeostatic cytokines.
Brain vascular endothelium promotes metastatic growth, invasion, altered architecture and function. Fewer vessels are more permeable and viable or hypoxic tumors produce VEGF by hypoxia-related gene expression, and tumor-associated stromal cells, e.g., fibroblasts, contribute to support metastases. Microglia and astrocytes may also be protective. Blood–brain barrier (BBB) histologically is composed of ECs, pericytes, astrocytic perivascular endfeet and makes an active process of blocking, because molecules can’t pass through tight junctions, but must pass through cells. An active transport system regulates the passage of small hydrophilic molecules, while large hydrophilic molecules are excluded or admitted via receptor-mediated endocytosis
Molecular mechanisms are common to various malignancies. Between these the most important are VEGF, hypoxia-related genes, WNT signaling, EGFR-related, Immune/inflammatory (TGF-β expression, CD4, NK cells, B7-H3).
Molecular mechanisms of particular interest for melanoma are STAT3 that regulates angiogenesis gene expression VEGF, and results in improved brain metastases with respect to primary tumor, so the level of gene expression are associated with potential for brain metastasis. Rx synergy of STAT3 block, and AKT that is hyperactivated in human brain metastases without PTEN loss promoted brain metastasis was modeled in genetically-engineered mouse. AKT may be activated by secreted product of astrocytes, and can have therapeutic implications
Nab-paclitaxel is the first nanotechnology-derived agent approved for the treatment of breast cancer. This formulation exhibits linear pharmacokinetics over a clinically relevant dose range 3, which means predictable drug exposure with dose modification. Compared with solvent-based paclitaxel, nab-paclitaxel exhibits linear pharmacokinetics, ~10-fold increase in Cmax and ~3-fold higher AUC (area under the curve) of unbound paclitaxel, potential binding to albumin-binding proteins and shows enhanced transport across endothelial cell monolayers, with a 33% higher paclitaxel concentration in tumor xenografts
Melanoma cells express high levels of SPARC (serum protein acidic and rich in cysteine), an albumin-binding protein that may facilitate delivery of nab-paclitaxel to tumor cells. SPARC, also known as Osteonectin and BM-40, is a 43-kDa glycoprotein that interacts with extracellular matrix proteins. Normal functions of SPARC may include tissue remodeling, angiogenesis, cell motility, mineralization of bone and cartilage, and embryonic development. SPARC is overexpressed in many cancers, especially in cells associated with the tumor stroma and vasculature, and it may play a role in tumor progression in different cancer types.
There is a direct correlation of SPARC expression and OS in patients with pancreatic cancer who received nab-paclitaxel plus gemcitabine, as evidenced in a Phase I/II Trial, with a median OS of 17.8 months in the high SPARC expression group
In phase I and II studies nab-paclitaxel produced promising efficacy, differently from cremophor-paclitaxel or docosahexaenoic acid-paclitaxel, that produced limited clinical benefit. The phase III trial randomized 514 pts with chemo-naïve metastatic cutaneous melanoma to be treated with nab-paclitaxel or dacarbazine. Primary efficacy endpoint was PFS while secondary efficacy endpoints were OS, ORR and DCR. PFS of nab-paclitaxel group resulted 4.8 months, while PFS of dacarbazine group resulted 2.5 months. The advantage was also obtained in OS with 12.8 months for nab-paclitaxel group
Nab-paclitaxel is the first taxane and first single-agent chemotherapy in 35 years that demonstrated superiority compared with dacarbazine with a near doubling of median PFS, a 44% improvement in disease control rate (DCR), and a trend towards OS benefit in chemo-naïve patients with metastatic melanoma. Future perspectives could consist in the combination of nab-paclitaxel with newly approved biologic/immunologic therapies (BRAF inhibitors in BRAF mutated patients or ipilimumab independently of BRAF mutational status).
Understanding how tumor rejection occurs under a given immunotherapy is one of the primary goals of clinical research in order to optimize existing immunotherapy and development of new therapies. Through multiple cancer immunotherapy molecular monitoring, a repetitive phenomenon has been observed that the activation of different factors as STAT1/IRF1, allograft inflammatory factor 1, IL-15, IL-6, Granzyme A, B, K, Perforin, CCL4 (MIP-1b), CXCL10/IP-10, CXCL9/Mig, and/or CCL5 are necessary for tumor rejection. Nevertheless, tumor rejection is a multifactorial process involving host’s genetic background, environmental/hidden factors and tumor genetics.
In order to dissect contribution of each components to the clinical outcome of melanoma patients undergoing adoptive T cell therapy TILs from 142 patients enrolled in adoptive T cell therapy trials, 112 parental melanoma metastases and 15 melanoma cell lines derived from melanoma metastases were analyzed. Global transcriptome analysis of the tumors sample using gene expression array analysis, micro RNA profiling as well as genome wide single nucleotide polymorphism (SNP) association analysis of advanced melanoma patients who achieved CR and of those that progressed (NR) was performed. The results demonstrate that TILs that lead to complete tumor regression in patients share a gene signature distinctive of memory cells. Pathway enrichment analysis revealed down regulation of genes associated with differentiation as well as enhanced proliferation function in CR compared to NR patients. The data underscore the notion that these T cells are less differentiated but have enhanced proliferative phenotype consistent with recently published data
Significant segregation of responders from non-responders was also observed based on the gene expression profile of the tumor cell lines, leading to the conclusion that immune responsiveness is at least in part dependent upon the biology of cancer cells
Comparison of the miR profile in complete responders versus nonresponders identified 13 distinct miRs between CR and NR, suggesting that clinical outcome can be regulated at the epigenetic level. Genome-wide scan analysis of germline SNPs based on allele frequency identified 5 loci in IRF5 gene with global significance that were associated with clinical outcome suggesting that genetic polymorphism is an important component determining tumor rejection.
Concluding, immune responsiveness is at least in part is dependent upon the genetic background of the host and the biology of cancer cells. The finding that adoptive T cells have enhanced proliferative capacity and can sustain the function of memory and effector T cell subsets has considerable implications for the design of T cell–based therapy to target intracellular pathogens and cancer.
Sentinel node biopsy has completely changed the surgical approach to melanoma patients, it has permitted to stage properly the patients and it has opened new ways of researches on the biology of tumor metastases. By the newer technologies a computer-assisted probe with adjustable collimation has been developed. By this machine, energy windows can be calibrated by computer, according to the energy spectrum emerging from each patient. In this way, the possibility to optimize photoelectric/compton efficiency reduces the dimensions of the smallest detectable node, possibly reducing the false negative sentinel lymph node biopsies and the possibility of errors of surgeons and nuclear radiologists. Conventional SPECT imaging is a Single Photon Emission Computed Tomography
Sentinel node (SN) is the first point of contact between tumor-associated antigens and the adaptive immune system, and is the site where dendritic cells (DCs), the professional antigen presenting cells (APCs), are expected to present tumor antigens to naive T-cells generating specific responses against tumors. SNs show down-regulation of APCs, because DCs show little or no formation of dendritic processes. Plasmacytoid dendritic cells (p-DCs) have been described in primary melanoma lesions and p-DCs represent the main source of IFN-α. There is a possible role of p-DCs in priming naive melanoma-specific CD8+ T-cells as well as in IFN production. The infiltration of p-DCs has a prognostic value in primary breast cancer, as its presence correlates with adverse outcome. p-DCs are rarely detected in lymph nodes of healthy subjects. p-DCs have been demonstrated in SN of melanoma patients and they accumulate around melanoma nests in situ, and their percentage increases in presence of melanoma metastasis
Electrochemotherapy (ECT) is a new therapy for cutaneous metastases that uses reduced doses of antineoplastic drug in association with electroporation to create a difference of potential in the cell membranes that opens the pores by which the chemotherapy agent enters inside the cell and gives a cytotoxicity-induced DNA break. Electrochemotherapy is effective, with 85% Objective Response (OR), 73.7% CR and 11.1% Partial Response (PR). The procedure is safe; the side effects are absolutely slights with only erythema, edema, necrosis, discromia and with no grade III-IV toxicity. Possible mechanisms of action are cytotoxicity on cancer cells that give necrosis and apoptosis of cancer cells by electric pulses. Immunological mechanisms could play a role, as 80% of tumors cures were achieved in immunocompetent mice and none in immunodeficient mice. Moreover, it has been demonstrated a reduction of epidermal LCs at biopsies performed at day 7 post-ECT, an increase of epidermal LCs at day 14 post-ECT, an increased amount of peri-tumoral dermal DCs CD1c+, HLA-Dr + post-ECT and the appearance of peri-tumoral CD83+ DCs in the dermis post-ECT
Liver metastases are a typical manifestation of progression of disease of uveal melanoma, but even cutaneous melanoma demonstrates predominant hepatic metastases in some cases. Liver anatomy is favorable to vascular isolation, as tumors receive the majority of their blood flow from the hepatic artery, and hepatic metastases are often the cause of death of the patient. Isolation and arterial perfusion of the liver eliminates or significantly reduces systemic toxicity, and allows higher doses of toxic chemotherapy. Isolated liver perfusion began in the 1960’s, using an open, operative technique. The advent of advanced vascular catheters and an extracorporeal venous hemofiltration system have allowed development of a minimally invasive, percutaneous technique. A phase I study of Hepatic Arterial melphalan Infusion and Hepatic Venous Hemofiltration using percutaneous catheters in patients with unresectable hepatic malignancies enrolled 12 patients treated at dose of 2.0 mg/kg then dose escalation (2.5, 3.0, 3.5 mg/kg)
Bridge between melanoma and colo-rectal cancer: biology of tumor microenvironment
Discovery of specific mutations leads to development of clinically useful inhibitors of critical signaling pathways but usually is limited to specific type of tumor. However, the importance of the immune response and the role of microenvironment in cancer development, progression and metastasis has been recognized as potential therapy target to improve cancer patients survival in many tumor types, including melanoma. For example, the critical role of the microenvironment in the growth of cancer is supported by the role and the therapeutic potential of angiogenesis. Current data indicate that secreted stromal factors that can mediate drug resistance and predict response to treatment based on microenvironmental factors could be shared between various tumors. Thus, promising studies of microenvironment and immune response in different type of tumors can benefit melanoma and
A lack of balance of host tumor interaction is due to numerous interactions and is mediated by at least three distinct escape mechanisms involving:1) alteration of genomic and phenotypic features of tumor cells themselves (expression of tumor antigens, somatic mutations, up-regulation of anti-apopototic molecules, 2) the tumor micromilieu (melanoma cells interact with the microenvironment in bidirectional manner through molecular signals that modulate malignant phenotype), as well as 3) immune cells that can either become immunosuppressed or reject the tumor cells. Cancer cells interact with stromal cells by production of certain factors affecting fibroblasts or ECs resulting in a cascade of events leading to progression For example, melanoma cells secrete growth factors such as FGF-β PDGF, and TGF-β that affect angiogenesis and stroma formation by inducing proliferation of fibroblasts and ECs. The melanoma cells can also produce VEGF resulting in ECs growth. In return, EC produce chemokines resulting in melanoma cell migration (e.g., CCR4) via receptors found on melanoma cells that can contribute to drug resistance to cancer therapy. HGF secretion by stromal cells was shown to be responsible for resistance to targeted therapy including BRAFi. The impact of interactions of tumor cells and stroma is increasingly recognized as important target to individualize therapy for melanoma patients.
An altered interplay between immune system and tumor cells towards inflammation that promotes carcinogenesis represents a major cause of tumor progression. This process includes downregulation of MHC molecules, secretion of immune suppressive cytokines (VEGF, IL-10 and TGF-β, recruitment of inhibitory immune cells (e.g., Treg and MDSCs) and others. Loss or downregulation of HLA class I antigens often occurs in tumor cells and is associated with disease progression, reduced survival of patients as well as lack of response to T cell-based immunotherapies. Although, metastasis expressing HLA class I antigens in melanoma patients undergoing immunotherapy can regress immunotherapies can partially generate HLA class I loss variants causing progression. The HLA class I abnormalities are mediated by an impaired expression and functional defect of distinct components of the antigen processing machinery (APM) resulting in a decreased HLA class I surface expression, an altered antigen repertoire as well as a reduced recognition by CD8+ cytotoxic T lymphocytes (CTL)
Targeting antigens expressed by tumors in their microenvironment was evaluated in a multi-step process that focused on patients with non small cell lung cancer (NSCLC) or prostate cancer who received autologous tumor vaccines. Protein arrays (ProtoArray) were used to detect the difference in pre- and post-treatment antibody responses. Since autologous tumor was not available for most patients, gene expression profiles of established cell lines were used. Studies with NSCLC patients exhibiting a CR (n = 3) or stable disease (n = 1) found a common pattern of antibody responses against antigens that were shared by the 13 NSCLC cell lines
Next a novel immunotherapy strategy employing autophagosome vaccines that contain defective ribosomal products (DRiPs) and short-lived proteins (SLiPs) was reviewed. DRiPs and SLiPs are the center of MHC class I antigen processing pathway, and link the immunosurveillance of viruses and tumors. DRiPs enable the immune system to rapidly detect alterations in cellular gene expression with great sensitivity. DRiPS and SLiPS are rapidly degraded by proteasome, transported to the endoplasmic reticulum, bound by MHC Class I and transferred to the cell surface where they are postulated to contribute the majority of peptides decorating the surface of tumor cells. Proteosome blockade shunts DRiPS and SLiPS to the autophagy pathway and by blocking a degradative step, autophagosomes can be harvested and used as a vaccine. In preclinical studies, an autophagosome vaccine was more therapeutic than a “Gold Standard” GVAX Vaccine in a 3 day-established 3LL tumor model. Additional studies documented that autophagosomes from one unique sarcoma can prime an immune responses against other independently-derived syngeneic sarcomas and can provide a significant level of protective immunity (challenged 14 days post vaccination) in 8 of 9 tumor combinations tested. In contrast, whole tumor cell vaccines only provided protection from a tumor challenge when the challenge was identical to the tumor used as a vaccine
For optimal therapeutic vaccination against cancer concentrated antigen delivery (DNA, RNA, SLP) with appropriate adjuvant is crucial. Synthetic vaccines allow rational vaccine design. Favoured cancer target antigens are involved in cancer initiation, progression and/or metastasis. Example: oncogenic proteins E6 and E7 of high risk HPV. Long peptide vaccines harboring both CD4 and CD8 T cell epitopes and requiring DC processing are efficient and were found capable of causing >50CR or PR in patients with high grade vulvar intraepithelial neoplasia (VIN), caused by HPV16
Malignant Mesothelioma (MMS) is a rapidly progressive lethal tumor and the incidence is steadily increasing worldwide. An actual “epidemic” is expected over the next 10–15 years. No standard treatment significantly improves prognosis of MMS patients. Median OS is 12 months (ranged from 6 to 18 months) from diagnosis. Survival in pretreated patients is even poorer. The second-line therapy is undefined and enrollment in clinical trial for fit patients is encouraged. Immune therapy can be considered a good option of treatment, considering evidence demonstrating that lymphocytic infiltration of pleural mesothelioma correlated with a better survival. Consistently, it has been observed that patients with high level of tumor infiltrating T cells have an overall survival much longer than the patients with a low rate. Along this line different immunotherapeutic agents have been tested in MMS patients but with disappointing results. Anti-CTLA-4 mAb potentiate the anti-tumor immune response. The anti-CTLA4 mAb ipilimumab significantly improves the survival of metastatic melanoma patients (20% long-survivors to 4-yrs) and it is currently in clinical development for other indications. The anti-CTLA-4 mAb tremelimumab has been extensively tested in different tumor types, and it is currently being investigated for different indications. Both ipilimumab and tremelimumab can induce durable stabilization and clinical response (even after initial disease progression).
The MESOT-TREM-2008 Study is a second line, single arm, phase II study with the anti-CTLA-4 mAb tremelimumab in chemotherapy-resistant advanced malignant mesothelioma
Melanoma comprises multiple clinical forms that arise and develop through different pathogenic pathways, and understanding pathophysiological implications of specific genes associated with metastatic lesions is crucial to identify new prognostic and therapeutic targets. Consistent with the contribution of VEGF and integrin VLA-4 to prometastatic effects of IL-18 in experimental melanoma models
Results of this study demonstrated the occurrence of metastatic lesions with and without IL-18-dependent genes, suggesting that human melanoma metastasis development occurred via inflammation dependent and independent mechanisms. Signature genes from melanoma cell lines with and without IL-18R/VEGF/VLA-4 phenotype may serve as functional biomarkers of melanoma predisposition to prometastatic effects of IL-18. In this regard, melanoma lesions overexpressing those specific genes from untreated (TGFBI, FEZ1, SLC20A1, POLM, GPKOW) and IL-18-treated (UBE2C, UMPS, IQCE, PFAS, PPAT, PTPLAD, ZBTB9) highly IL-18R/VEGF/VLA-4-expressing melanomas, may be susceptible to prometastatic effects of IL-18, and therefore, should not be treated with this cytokine and other immunotherapies increasing inflammation. Conversely, those genes from highly (CD52, PRSS23, TMEM42) and low (CAPN3, A2M, ARID4A, CNOT6L, CYB561) IL-18R/VEGF/VLA-4 expressing melanomas not expressing IL-18-dependent genes, may be helpful to detect IL-18-unresponsive melanoma lesions, and therefore, better candidates for immunotherapy
Reported signature genes allowed for first time the classification of metastatic melanoma lesions according to IL-18R/VEGF/VLA-4 phenotype and IL-18-dependent gene expression. These genes may have diagnostic value as pathogenic biomarkers and therapeutic targets of melanoma predisposition to prometastatic effects of IL-18. At present, it is unknown if IL-18R/VEGF/VLA4 phenotype is necessary and sufficient for the determinism of metastasis in melanoma patients. However, many of the genes associated to IL-18R/VEGF/VLA4 phenotype are candidates for involvement in pathogenic mechanisms of melanoma metastasis based on their known function in melanoma and other malignancies. Therefore, these genes may also be a source of potential targets for the specific treatment of metastases developed via IL-18-dependent and independent mechanisms.
Traditionally, the anatomic extent of the tumor burden in colorectal cancer (CRC) and other type of solid tumors has been the most important prognostic factor. Data on the tumor burden (T-stage), combined with the presence of cancer cells in draining and regional lymph nodes (N-stage) and evidence of metastases (M-stage), amalgamate to provide tumor staging (AJCC/UICC-TNM classification). TNM stages estimate patient post-operative outcome and the rationale for adjuvant therapy. Despite the prognostic power of this staging system, it is becoming recognized within the cancer community that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Advances in this field have alluded to the importance of the immune prevalence within the tumor microenvironment. Histological analysis of human tumors, in particular CRC, has highlighted the importance of the combination of immune variables. We have previously described these major immune parameters, associated with survival in colon cancer, as the “immune contexture”
Derived from the immune contexture, a simple and powerful immune-classification has been termed the “Immunoscore”. The Immunoscore (I) is based on the numeration of two lymphocyte populations (CD3/CD45RO, or CD3/CD8 or CD8/CD45RO) quantified within the center of the tumor (CT) and invasive margin (IM). These parameters provide a scoring system ranging from Immunoscore 0 (I0), which has low densities of both cell types in both regions; to Immunoscore 4 (I4), having high densities of both cell populations in both regions. The prognostic value of using these immune criteria was demonstrated in patients with early stage CRC (AJCC/UICC TNM stage I-II CRC) to predict survival and relapse. The five Immunoscore groups were associated with dramatic differences in DFS and OS (P < 0.0001)
In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. The working group, composed of international expert pathologists, oncologists and immunologists, identified a strategy for the organization of worldwide participation by various groups for the validation of the Immunoscore. The purposes of the Immunoscore worldwide task force are, to demonstrate the feasibility and reproducibility of the Immunoscore, to validate the major prognostic power of the Immunoscore in routine for patients with colon cancer stage I/II/III, and to demonstrate the utility of the Immunoscore to predict stage II colon cancer patients with high risk of recurrence. Twenty-three (23) international expert centers agreed to participate in this visionary enterprise. These participants represent 23 Centers Worldwide from 17 countries including Asia, Europe, North America, Australia, and Middle East. The outcome of this international validation effort may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated as TNM-I (TNM-Immune).
Competing interests
PAA has consultant and advisory role for Bristol Myers Squibb, Merck Sharp & Dohme, Roche-Genenetech, Glaxo Smith-Kline, Amgen, Celgene, Novartis. He received honoraria from Bristol Myers Squibb, Merck Sharp & Dohme, Roche-Genenetech. He received research fund from Bristol Myers Squibb.
AMG has no competing interest.
NA has no competing interest.
LB has no competing interest.
LC has no competing interest.
NC has no competing interest.
AC is full time employee of Nodality Inc., salary and shares.
MDV received Research Funding from Celgene, Novartis, Roche, and Glaxo SmithKline.
AME participated to advisory boards for BMS, GSK, MedImmune, MSD.
MF participated to advisory board for Amgen; he was also consultant for Delcath.
SF has no competing interest.
BAF is a co-founder of and has an ownership interest in UbiVac, the company developing autophagosome vaccine technology.
TG participated to advisory board from GSK-Bio, Roche-Genentech, Merck, BMS, Abbvie; he is co-founder of Jounce; he received research support from GSK-Bio, Eisai, Roche-Genentech, BMS, Curetech, Morphotek, Incyte.
JG is consulting for Ventana-Roche; he participated to advisory board for Jounce; he has a research contract with MedImmune.
SG is an inventor on patents/patent application regarding the NY-ESO-1 antigen, and Ludwig Institute for Cancer Research has licensed such patents.
HG has an advisory role for Roche, Glaxo, BMS, and MSD.
MKS owns stock in Melanoma Diagnostics, Inc.
HLK has research support, participated to advisory board and received honoraria from Amgen.
JL participated to advisory boards from Pfizer, Novartis, Aveo, BMS, GSK; he received research funding from Novartis and Pfizer.
RL has no competing interest.
AM has no competing interest.
KM received clinical research support from Genentech/Roche, BMS, GSK, Merck, Altor, Novartis, Prometheus.
CM is CSO of ISA Pharmaceuticals, trying to market SLP vaccines, for which he receives a salary and has stock appreciation rights.
GMA has research support from Pfizer, Novartis and Millennium.
GP has no competing interest.
IP has no competing interest.
AR has been a consultant for Amgen, GSK, Roche, Novartis with the honoraria paid to UCLA.
BS has no competing interest.
JS participated to advisory board for BMS, GSK, Roche-Genentech with honorarium.
PS is an employee of GSK Vaccines.
AAT has participated to advisory board from Merck, Genentech, Prometheus, AstraZeneca; he received research funding (contracted with the University of Pittsburgh) from Merck, BMS, Prometheus, Pfizer, Novartis, Amgen.
GT has no competing interest.
FVV has no competing interest.
EW has no competing interest.
GC has no competing interest.
NM has no competing interest.
FMM has no competing interest.
MT has no competing interest.
Authors’ contributions
PAA, AMG, and MT prepared the manuscript collaboratively with input and review by all co-authors. All authors read and approved the final manuscript.
All the data and results reported into the manuscript are compliant with the Helsinki Declaration and the international guidelines regarding any experimental research.